Hindawi Publishing Corporation

Journal of Pathogens
Volume 2016, Article ID 3264070, 5 pages
http://dx.doi.org/10.1155/2016/3264070

Research Article
In Vivo Antimalarial Activity of Annona muricata Leaf Extract
in Mice Infected with Plasmodium berghei

Voravuth Somsak, Natsuda Polwiang, and Sukanya Chachiyo

Department of Clinical Chemistry, Faculty of Medical Technology, Western University, Kanchanaburi 71170, Thailand

Correspondence should be addressed to Voravuth Somsak; voravuthsomsak@gmail.com

Received 6 November 2015; Revised 5 March 2016; Accepted 6 March 2016

Academic Editor: Alexander Rodriguez-Palacios

Copyright 2016 Voravuth Somsak et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Malaria is one of the most important infectious diseases in the world. The choice for the treatment is highly limited due to drug
resistance. Hence, finding the new compounds to treat malaria is urgently needed. The present study was attempted to evaluate
the antimalarial activity of the Annona muricata aqueous leaf extract in Plasmodium berghei infected mice. Aqueous leaf extract
of A. muricata was prepared and tested for acute toxicity in mice. For efficacy test in vivo, standard 4-day suppressive test was
carried out. ICR mice were inoculated with 107 parasitized erythrocytes of P. berghei ANKA by intraperitoneal injection. The
extracts (100, 500, and 1000 mg/kg) were then given orally by gavage once a day for 4 consecutive days. Parasitemia, percentage
of inhibition, and packed cell volume were subsequently calculated. Chloroquine (10 mg/kg) was given to infected mice as positive
control while untreated control was given only distilled water. It was found that A. muricata aqueous leaf extract at doses of 100,
500, and 1000 mg/kg resulted in dose dependent parasitemia inhibition of 38.03%, 75.25%, and 85.61%, respectively. Survival time
was prolonged in infected mice treated with the extract. Moreover, no mortality to mice was observed with this extract up to a
dose of 4000 mg/kg. In conclusion, the A. muricata aqueous leaf extract exerted significant antimalarial activity with no toxicity
and prolonged survival time. Therefore, this extract might contain potential lead molecule for the development of a new drug for
malaria treatment.

1. Introduction important for killing of malaria parasite. The use of medicinal

Malaria, a tropical parasitic disease, is one of the most impor-
tant infectious diseases in the world. Estimated 3.3 billion of
the total world population live in areas with malaria risk and
an estimated death of 660,000. Africa is the most affected
continent, accounting for about 90% of all malaria deaths [1].
This disease is caused by Plasmodium species and transmitted
by the bite of female Anopheles mosquito. The global strategy
for malaria mainly focuses on case management through pro-
vision of drugs capable of reducing or eliminating parasites.
However, multiple antimalarial drug resistant Plasmodium
parasites and the emergence of insecticide resistant Anopheles
mosquitoes are causing not only the spread of malaria to new
areas but also its reemergence in areas where it had previously
been eradicated [2]. In addition, chloroquine resistance
now occurs throughout the whole world [3]. Therefore, the
search for new antimalarial, either synthetic or natural, is
plant extracts for the treatment of malaria has a long and
successful tradition [4]. For example, quinine was isolated
from Cinchona and artemisinin from Qinghaosu [5]. This
explains why a lot of current researches focus on natural and
plant-derived products as they can be sourced easily, can be
locally available, and can be selected on the basis of their
pharmacological use [6, 7].
Annona muricata, a member of the Annonaceae family, is
now widely distributed throughout tropical and subtropical
parts of the world such as South and North America, Africa,
Asia, and Southeast Asia including Thailand [8]. A. muricata
extract presents antioxidant, anti-inflammation, antimicro-
bials, antiparasitic, antidiabetes, antihyperlipidemia, hepato-
protective, and anticancer activities [914]. Extensive phy-
tochemical evaluations of A. muricata extract have shown
the presence of various compounds, including alkaloids,
polyphenols, flavonoids, essential oils, cyclopeptides, and
2 Journal of Pathogens
kaempferol [15]. Moreover, A. muricata has been shown to
be a generally rich source of acetogenin compounds that are
active compounds in this plant extract [8, 15]. Furthermore,
the leaf extract of A. muricata was assayed against P. fal-
ciparum and showed promising antimalarial effect [12, 16].
However, antimalarial activity of this plant extract against
P. berghei infected mouse model has not yet been reported.
Hence, the present study aimed at evaluating the in vivo
antimalarial activity of A. muricata aqueous leaf extract
against P. berghei infected mice.
2. Materials and Methods
2.1. Preparation of Aqueous Leaf Extract. The dried leaves
of Annona muricata were purchased from Royal Project
Foundation shop, Chiang Mai, Thailand. The dried leaves
were ground using electric blender, and extraction was then
carried out by dispersing 10 g of the dried powdered plant
material in 100 mL of distilled water (DW). Microwave
method was used at 360 W for 5 min and incubated at room
temperature for 24 h [17]. Filtration was then performed
through Whatman number 1 filter paper and followed by
lyophilization. Aqueous leaf extract of A. muricata was stored
at 20 C until used. Before experiments, the extract was then
dissolved in DW at appropriate doses.
2.2. Acute Toxicity Test. Acute toxicity of A. muricata aqueous
leaf extract was carried out using modified Lorkes method
[18]. Fifteen female ICR mice were randomized into 5 groups
of three mice each and were then given orally with 100, 500,
1000, 2000, and 4000 mg/kg. The mice were observed for
signs of toxicity such as paw licking, salivation, stretching of
the entire body, weakness, sleep, respiratory distress, coma,
and death in the first four hours and subsequently daily for
7 days. The oral median lethal dose was calculated using the
following formula:
LD50
(1)
= (Minimum toxic dose Maximum tolerate dose).
2.3. Experimental Mice. Pathogen-free (bacteria, fungi, and
parasites), 4-week-old female ICR (Imprinting Control
Region) mice weighting 2530 g were obtained from the
National Laboratory Animal Center, Mahidol University,
Bangkok, Thailand, and kept for at least one week with sterile-
filtered tap water and pellet diet (CP diet 082, Perfect Com-
panion Company, Bangkok, Thailand) ad libitum. Experi-
ments were started in 5-6-week-old mice. Procedures of the
animal experiments were ratified by the Ethical Committee
on Animal Experimentation, Faculty of Medical Technology,
Western University.
2.4. Rodent Malaria Parasite. Plasmodium berghei ANKA
(PbANKA) was used. Frozen parasites from stock were
Parasitemia of untreated group Parasitemia of extract treated group
% inhibition =
Parasitemia of untreated group
passaged at least once through ICR mice before experiments.
Parasitemia was daily monitored by microscopy of Giemsa
stained thin blood smear. The percentage of parasitemia (%
parasitemia) was calculated using the formula
% parasitemia
Number of parasitized erythrocytes (2)
= 100.
Number of erythrocytes
When the parasitemia showed 1520%, infected mouse
blood was then collected by cardiac puncture and suspended
in phosphate buffer saline (PBS). Infection for experiments
was carried out by intraperitoneal (IP) injection of approxi-
mately 107 parasitized erythrocytes in mice.
2.5. Determination of Mean Survival Time. Mortality was
monitored daily and the number of days from the time of
inoculation of the parasite up to death was recorded for each
mouse. The mean survival time (MST) was calculated as
follows:
MST
(3)
Sum of survival time of all mice in a group
= (days) .
Total number of mice in that group
2.6. Determination of Packed Cell Volume. The packed cell
volume (PCV) of each mouse was measured by collecting
tail blood into heparinized hematocrit tubes, sealed with
Critoseal, and placed in a hematocrit centrifuge at 10,000 rpm
for 5 min. PCV was then read using hematocrit reader
according to the following formula:
Volume of packed erythrocytes
PCV = 100. (4)
Total volume of blood
2.7. Standard Antimalarial Drug. Chloroquine (CQ) was
used as standard antimalarial drug in this study. The drug at
chosen dose was freshly prepared in DW and administered
orally by gavage. Drug dose, expressed in mg/kg of body
weight, was adjusted at the time of administration according
to the weight of the mice.
2.8. Suppressive Test. The standard 4-day suppressive test
against PbANKA infection in mice was employed [19]. Nave
ICR mice were inoculated by IP injection of 107 parasitized
erythrocytes. The infected mice were randomly divided into
5 groups of 5 mice per group and treated for 4 consecutive
days with 100, 500, and 1000 mg/kg of extract orally by
gavage. Two control groups were used: the positive control
was treated daily with 10 mg/kg of CQ while the untreated
group was given DW. On day 5 of experiment, parasitemia
and percentage of inhibition were calculated according to the
following formula:
100. (5)
Journal of Pathogens 3

Table 1: Effect of A. muricata aqueous leaf extract on parasitemia and mean survival time.

Treatment Dose (mg/kg) % parasitemia % inhibition Mean survival time

100 34.02 1.5 38.03 18.32 0.84
A. muricata 500 13.59 1.8 75.25 24.79 1.76
1000 7.9 0.2 85.61 28.75 1.37
Chloroquine 10 0.52 0.05 99.05 30.33 1.61
Distilled water 1 mL 54.9 4.39 0 7.79 0.73

< 0.05, < 0.01, and < 0.001, compared to untreated control. Parasitemia and mean survival time were expressed as mean SEM, = 5.
Chloroquine: positive control; distilled water: untreated control.

2.9. Statistics. The one-way ANOVA was used to analyze and
compare the results at a 95% confidence. Values of < 0.05
were considered significant. Results were expressed as mean
standard error of mean (SEM).
3. Results
3.1. Acute Toxicity. The results of the acute toxicity evaluation
of A. muricata aqueous leaf extract showed no remarkable
behavioral changes in the extract administered mice. No
mortality occurred within the observation period of 7 days.
However, behavioral signs of toxicity were observed in mice
given 4000 mg/kg which include paw licking, salivation,
and stretching and reduce activity. There was however no
mortality at all the doses used. The oral median LD50 was
estimated to be 2500 mg/kg.
3.2. Effect of A. muricata Aqueous Leaf Extract on Parasitemia
and Mean Survival Time. The results of the standard 4-day
suppressive test of the A. muricata aqueous leaf extract on
parasitemia and survival time in PbANKA infected mice
were summarized in Table 1. The A. muricata aqueous leaf
extract presented dose dependent antimalarial effect against
PbANKA infection in mice and caused a significant ( <
0.05) inhibition when compared to the untreated group. The
highest inhibition (85.61%) was observed with the dose of
1000 mg/kg. However, the mice were not completely cured
from the infection in all treatment doses but did significantly
( < 0.05) prolong the mean survival time at all dose levels.
3.3. Effect of A. muricata Aqueous Leaf Extract on Packed Cell
Volume. The effect of A. muricata aqueous leaf extract on
PCV on day 0 and day 4 comparison was indicated in Table 2.
The extract showed protective effect on PCV reduction in
dose dependent manner, although the dose of 100 mg/kg
resulted in significant ( < 0.05) reduction of PCV.
4. Discussion
Medicinal plants contain active constituents that have poten-
tial for medicinal use and pharmaceutical drug companies
make use of these plants [7, 20, 21]. The extract of A. muricata
was reported to contain different classes of metabolites
such as tannins, alkaloids, flavonoids, polyphenols, saponins,
Table 2: Effect of A. muricata aqueous leaf extract on packed cell
volume.
Packed cell volume
Treatment Dose (mg/kg)
Day 0 Day 4
100 53.24 2.73 40.14 3.26
A. muricata 500 50.56 3.12 48.22 3.18
1000 53.71 2.59 52.15 1.76
Chloroquine 10 51.55 3.14 52.47 2.27
Distilled water 1 mL 52.15 3.25 38.26 2.31
< 0.05 and < 0.01, compared to day 0. Packed cell volume was
expressed as mean SEM, = 5. Chloroquine: positive control; distilled
water: untreated control.
diterpenoids, essential oils, kaempferol, and acetogenin com-
pounds [8, 15, 22, 23]. Diterpenoids, flavonoids, polyphenols,
saponins, alkaloids, kaempferol, and acetogenin were known
to have antimalarial activity [2427]. Moreover, antioxidant
effect in this plant may also contribute to the antimalarial
activity. It has been reported that antioxidant activity can
inhibit heme polymerization as heme has to be oxidized
before polymerization, and the unpolymerized heme is very
toxic for malaria parasite [28, 29]. In addition, these active
compounds in this plant extract may be acting singly or
in synergy with one another to exert the observation anti-
malarial activity. All doses of the extract displayed significant
and dose dependent inhibition in comparison with untreated
control. So, the activity may be due to the individual or
synergistic effect of the metabolites found in the extract like
alkaloid, flavonoids, polyphenols, saponins, kaempferol, and
acetogenin. It is finding similar with the previous study that
showed antimalarial activity against P. falciparum [12, 16].
Scoring system used to assess signs of toxicity in mice
has not been performed. Physical observation for signs of
toxicity in mice has just been done. From this study,
all mice treated with this extract were normal in physical
activities and survived. Blood biochemical tests in order to
ensure the toxicity have not been carried out. However, in
this observation signs of toxicity have been used in other
studies [18]. Interestingly, A. muricata aqueous leaf extract
did not show mortality within 7 days up to a dose of
4000 mg/kg indicating that this extract is safe. In general, if
the lethal dose of the test compound is 3 times more than
the minimum effective dose, the compound is considered a
good candidate for further studies [30, 31]. The result is in
4 Journal of Pathogens
agreement with other studies done on the same plant with
crude extracts, indicating that no death was observed with
different dose levels, 1000, 3000, and 4000 mg/kg [12, 16].
However, abnormalities like depression, weakness, and rough
hair coat were observed with 4000 mg/kg aqueous crude leaf
extract of A. muricata.
The A. muricata aqueous leaf extract prolonged the MST
of the infected mice indicating that it suppressed PbANKA
and reduced the overall pathologic effect of the parasite
on the infected mice. Moreover, infected mice suffer from
anemia because of erythrocyte destruction, either by malaria
multiplication or by spleen reticuloendothelial cell action
[32]. In this study, the extract showed protection against PCV
reduction as compared to day 0 indicating that antioxidant
and antimalarial effects of this extract may play a critical
role. So, the A. muricata aqueous leaf extract inhibited and
reduced parasitemia in mouse followed by protection of PCV
reduction resulting to prolong the MST of the infected mice.
5. Conclusions
It is evident based on these findings that the oral administra-
tion of A. muricata aqueous leaf extract (1001000 mg/kg) to
mice for 4 days significantly reduced parasitemia of PbANKA
in experimental mice with nontoxicity. The implication of
this finding is that A. muricata aqueous leaf extract possesses
potent antimalarial effect and may therefore serve as potential
sources of safe, effective, and affordable antimalarial drugs.
The displayed high in vivo antimalarial property and lack of
toxic effect render A. muricata a candidate for the bioassay-
guided isolation of compounds which could develop into
new lead structures and candidates for drug development
programs against human malaria.
Competing Interests
The authors have declared that they have no competing
interests.
Acknowledgments
This work was supported by the Research and Academic
Science Service Division, Western University. Dr. Chairat
Uthaipibull at BIOTEC, NSTDA, and Associated Professor
Dr. Somdet Srichairatanakool at Department of Biochem-
istry, Faculty of Medicine, Chiang Mai University, are spe-
cially thanked for their help in discussion and suggestion of
malaria infection in vivo.
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